1. Field Of The Invention
The present invention relates to recombinant DNA technology. It appears especially useful for improving the nodulation (and thus nitrogen fixation) capability of plants.
2. Description Of The Art
Root nodule Rhizobium bacteria are responsible for symbiotic nitrogen fixation in the nodules of certain plants (e.g. legumes). Where natural bacterial activity is ineffective, the plants must rely on the existing nitrogen in the soil or on fertilizers. Where the former occurs, the quality of the soil is reduced. Where the latter occurs, the cost to the farmer (and ultimately the public) can be substantial. Further, the use of fertilizers often raises environmental concerns.
It is now known that the presence of certain "inferior" strains of Rhizobium in soil can depress the productivity of not only other natural bacteria, but also of "superior" bacteria added by inoculation of seeds. This can frustrate attempts to inoculate seeds prior to planting or to inoculate roots during plant growth. When inoculation has been successful, it is usually because the indigenous bacterial populations have been small.
Many investigators have studied the factors involved in determining nodule occupancy by strains of Rhizobium. See e.g. D. Dowling et al., 40 Annu. Rev. Microbiol. 131-157 (1986) (the disclosure of this article and of all other articles referred to herein are incorporated by reference as if fully set forth). Despite this work, no solutions to the above described Rhizobium competition problem have been developed.
In E. Triplett et al., 85 Plant Physiology 335-342 (1987) and 11th North American Rhizobium Conference Abstract GP4 (1987), my laboratory reported on the fact that the Rhizobium leguminosarum bv. trifolii bacterial strain T24 appeared to have genes in its coding responsible for a suppressor of other Rhizobium (I named the substance trifolitoxin) and other genes coding for T24's own resistance to trifolitoxin's effects. Unfortunately, I also have found that trifolitoxin production by transconjugant bacterial cells that I had constructed was readily lost in the absence of tetracycline. Thus, the earlier Rhizobium transconjugants were not likely to be able to effectively limit nodulation by trifolitoxin-sensitive indigenous strains of Rhizobium under agricultural conditions (where tetracycline application is impractical).
It was therefore desired to more specifically isolate and characterize the genes responsible for the T24 suppressor and resistance characteristics and use information developed therefrom to find a means for stably inserting such genes in the genome of "superior" Rhizobium so as to ultimately lead to a Rhizobium that can form effective nodules notwithstanding the presence of indigenous strains.